A method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria using ND-EESI-MS

Abstract

The present invention belongs to the field of detection technology, and particularly relates to a method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS, comprising: preparing antibacterial gold nanoparticles, determining the minimum inhibitory concentration of antibacterial gold nanoparticles to the bacteria to be tested, adding or not adding antibacterial gold nanoparticles to the bacterial solution to be tested, culturing, centrifuging and collecting the supernatant to obtain a sample to be tested and a control sample, neutral desorption electrospray extraction ionization mass spectrometry detection, analyzing the differential metabolites of the sample to be tested that are significantly reduced relative to the control sample, supplementing the differential metabolites to the bacterial solution to be tested containing antibacterial gold nanoparticles, and verifying whether the differential metabolites are the key antibacterial targets of the antibacterial gold nanoparticles. The present invention explores the molecular mechanism of antibacterial nanomaterials by detecting the changes in bacterial metabolites under the dynamic interaction between antibacterial gold nanoparticles and bacteria, and lays a foundation for screening antibacterial agents and guiding the development of new drugs.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and specifically relates to a method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria using ND-EESI-MS. Background Art

[0002] The problem of bacterial resistance is becoming increasingly serious, which has weakened the efficacy of traditional antibiotics, leading to rising treatment costs and prolonged recovery periods. The surge in multidrug-resistant (MDR) bacterial infections around the world poses a serious threat to public health, prompting researchers to seek innovative antibacterial strategies. Among them, gold nanomaterials (AuNPs) have attracted attention due to their unique physical and chemical properties, showing advantages such as efficient preparation, good antibacterial properties, high biosafety, and low resistance to inducing drug resistance. A series of innovative research works in the early stage have preliminarily confirmed that antibacterial AuNPs can destroy cell wall structure, interfere with bacterial metabolism, and inhibit bacterial proliferation. However, there are still many unclear points about the dynamic interaction between antibacterial AuNPs and bacteria and the antibacterial mechanism, which need to be further explored. Although traditional microscopes can observe the adsorption, aggregation and bacterial morphological changes of AuNPs on the bacterial surface, they are limited by the single observation state, limited resolution and complex sample pretreatment, making it difficult to obtain accurate antibacterial mechanisms. Therefore, it is urgent to develop new methods to deeply explore the antibacterial mechanism of gold nanomaterials.

[0003] Neutral desorption electrospray extraction ionization mass spectrometry (ND-EESI-MS) analysis technology, combined with electrospray extraction ionization and mass spectrometry analysis, can monitor material changes at the molecular level. It can provide high-sensitivity and high-resolution analysis results without complex pretreatment, and is suitable for quantitative analysis. Domestic and foreign scholars have made some explorations in the detection of complex samples using electrospray extraction ionization mass spectrometry technology. In the field of food safety, a Chinese patent (Patent Publication No.: CN104897767A) discloses a neutral desorption-electrospray extraction ionization mass spectrometry method for rapid detection of poisoned dead ticks in honey, a Chinese patent (Patent Publication No.: CN103822964A) discloses a neutral desorption-electrospray extraction ionization mass spectrometry method for direct detection of chloramphenicol in honey, and a Chinese patent (Patent Publication No.: CN105445360A) discloses a neutral desorption electrospray extraction ionization mass spectrometry method for rapid detection of dichlorvos in honey. The above inventions do not require pretreatment of the test samples, and can achieve rapid detection and identification of toxic components in honey, thereby improving the defects of existing analytical technologies such as complex and time-consuming operation steps. In the field of life sciences, a Chinese patent (Patent Publication No.: CN111122691A) discloses a method for detecting Arabidopsis metabolites under salt stress using the ND-EESI-MS method, performing in situ live non-destructive analysis of Arabidopsis leaves under salt stress, quickly obtaining information on changes in components in leaves under salt stress, and detecting Arabidopsis metabolites under salt stress. At the same time, a Chinese patent (Patent Publication No.: CN112198214A) discloses an electrospray extraction ionization mass spectrometry detection system, which quickly detects components in exhaled gas and combines artificial intelligence methods and algorithms to determine the disease status. The above patents solve the technical problems of complex detection operation, long detection time, and high detection cost. However, electrospray extraction ionization mass spectrometry technology is rarely used in the field of nano-antibacterial, and there are no reports on the study of the interaction mechanism between nanomaterials and bacteria without the need for complex sample pretreatment. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria using ND-EESI-MS.

[0005] The present invention provides a method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS, comprising the following steps:

[0006] S1, preparing antibacterial gold nanoparticles, and determining the minimum inhibitory concentration of the antibacterial gold nanoparticles to the bacteria to be tested;

[0007] S2, preparing the bacterial solution to be tested;

[0008] S3, adding the antibacterial gold nanoparticles to the bacterial solution to be tested, culturing for 3-4 hours, centrifuging and collecting the supernatant to obtain a sample to be tested, wherein the concentration of the antibacterial gold nanoparticles in the bacterial solution is ≥ the minimum inhibitory concentration; centrifuging the bacterial solution to be tested that has not been treated with antibacterial gold nanoparticles, collecting the supernatant to obtain a control sample; performing neutral desorption electrospray extraction ionization mass spectrometry detection on the sample to be tested and the control sample;

[0009] S4, Neutral desorption electrospray extraction ionization mass spectrometry detection:

[0010] The ion source of the neutral desorption electrospray extraction ionization mass spectrometer was set to positive ion mode, and the parameters were as follows: the ion source used methanol as the extractant, the extractant passed through a quartz capillary, and N2 was used as the spray gas flow; a 2-5 kV positive high voltage was applied to the steel needle of the injection needle, and under the action of the electric field, charged droplets carrying the analyte were generated at the front end of the sample, and the charged droplets were desolvated to obtain gaseous analyte ions, which were introduced into the mass spectrometer for detection; in the collision-induced dissociation CID experiment, the parent ion isolation width was 1.6-2.0 Da, the collision energy was 20%-40%, and other conditions were automatically optimized by the system;

[0011] S5. Quantitatively analyze the differential metabolites in the test sample relative to the control sample, and verify whether the differential metabolites are the key antibacterial targets of the antibacterial gold nanoparticles by supplementing the differential metabolites into the test bacterial solution containing the antibacterial gold nanoparticles.

[0012] Furthermore, the antibacterial gold nanoparticles are gold nanoparticles modified with p-aminophenylboronic acid, and the preparation method thereof is as follows:

[0013] Dissolve p-aminophenylboronic acid, chloroauric acid trihydrate, triethylamine and Tween 80 in water and mix well to obtain a mixed solution;

[0014] Then, sodium borohydride is dissolved in water to obtain a sodium borohydride solution; the sodium borohydride solution is then added dropwise to the mixed solution, stirred, and when the color of the solution in the bottle turns brown, reacted in an ice water bath for 2-2.5 hours to obtain the antibacterial gold nanoparticles.

[0015] Furthermore, the mass volume ratio of the deionized water, p-aminophenylboronic acid, chloroauric acid trihydrate, triethylamine and Tween 80 is 8-12 mL: 6-8 mg: 18-20 mg: 45-55 μL: 26-32 mg.

[0016] Furthermore, the mass volume ratio of the deionized water and sodium borohydride is 1-3 mL: 5-9 mg.

[0017] Furthermore, the bacteria to be tested include drug-resistant Klebsiella pneumoniae and Klebsiella pneumoniae.

[0018] Furthermore, the differential metabolites include one or more of adenine, serine, homoserine, isoleucine, putrescine, γ-aminobutyric acid, arginine, spermidine, methionine and spermine.

[0019] Further, the flow rate of the extractant is 9-12 μL / min.

[0020] Furthermore, the spray air pressure is 0.5-0.6 MPa.

[0021] Furthermore, the quartz capillary temperature is 190-210°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention introduces antibacterial gold nanoparticles into bacteria, interacts with the bacteria, obtains differential metabolites of the bacteria, and then uses neutral desorption electrospray extraction ionization mass spectrometry (ND-EESI-MS) analysis technology to sensitively detect the differential metabolites of the bacteria. Without the need for complex sample pretreatment, the differential metabolites (biomarkers) of the bacteria are detected to determine whether the antibacterial gold nanoparticles have antibacterial activity, thereby determining whether the differential metabolites are the key antibacterial targets of the antibacterial gold nanoparticles, providing a new and effective tool for the clinical diagnosis of bacterial infections and public health care.

[0024] (2) The present invention utilizes neutral desorption electrospray extraction ionization mass spectrometry (ND-EESI-MS) analysis technology to monitor the dynamic changes of substances at the molecular level, providing theoretical and technical support for the study of the interaction mechanism between nanomaterials and bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The figure shows the antibacterial properties of A-AuNPs and AuNPs of the present invention; in the figure, A is the minimum inhibitory concentration (MIC) of A-AuNPs and AuNPs against different bacteria; B is a scanning electron micrograph of the destruction of bacterial structure by A-AuNPs.

[0027] Figure 2 Schematic diagram of the ND-EESI device of the present invention.

[0028] Figure 3This is the optimization diagram of ND-EESI-MS experimental parameters. In the figure, A is the flow rate of the extractant, B is the temperature, C is the voltage, and D is the nitrogen pressure.

[0029] Figure 4 Diagram of extractant optimization for ND-EESI-MS experiment.

[0030] Figure 5 This is the Kp difference ion analysis diagram before and after A-AuNPs treatment of the present invention; in the figure, A is the principal component analysis of the difference ions; B is the orthogonal partial least squares discriminant analysis; and C is the projection variable importance value.

[0031] Figure 6 This is the differential ion analysis diagram of MDRK.p before and after A-ANPs treatment of the present invention; in the figure, A is the principal component analysis of the differential ions; B is the orthogonal partial least squares discriminant analysis; and C is the projection variable importance value.

[0032] Figure 7 The secondary spectra of 10 differential compounds of the present invention are identified; in the figure, A is adenine, B is serine, C is isoleucine, D is homoserine, E is arginine, F is γ-aminobutyric acid, G is methionine, H is putrescine, I is spermidine, and J is spermine.

[0033] Figure 8 The linear relationship between the concentrations of the 10 differential compound substances of the present invention and the bacterial concentration is constructed; in the figure, A is adenine, B is serine, C is isoleucine, D is homoserine, E is arginine, F is γ-aminobutyric acid, G is methionine, H is putrescine, I is spermidine, and J is spermine.

[0034] Fig. 9 This is a graph showing the changes in Kp of 10 different compounds over time during continuous treatment of A-AuNPs (10 μg / mL) of the present invention. In the graph, A is adenine, B is serine, C is isoleucine, D is homoserine, E is arginine, F is γ-aminobutyric acid, G is methionine, H is putrescine, I is spermidine, and J is spermine.

[0035] Fig.10 This is a graph showing the changes in Kp of 10 different compounds over time during continuous treatment of AuNPs (10 μg / mL) according to the present invention; in the graph, A is adenine, B is serine, C is isoleucine, D is homoserine, E is arginine, F is γ-aminobutyric acid, G is methionine, H is putrescine, I is spermidine, and J is spermine.

[0036] Fig.11This is a graph showing the growth changes of bacteria in which three representative substances and a mixture thereof are supplemented into damaged bacteria induced by A-AuNPs (10 μg / mL) according to the present invention; in the graph, A is supplemented with homoserine, B is supplemented with spermidine, C is supplemented with adenine, and D is supplemented with a mixture of homoserine, spermidine and adenine.

[0037] Fig.12 This is a graph showing the changes in 10 different compounds over time in the continuous treatment of A-AuNPs (10 μg / mL) in MDRK.p of the present invention; in the graph, A is adenine, B is serine, C is isoleucine, D is homoserine, E is arginine, F is γ-aminobutyric acid, G is methionine, H is putrescine, I is spermidine, and J is spermine.

[0038] Fig.13 This is a graph showing the changes in 10 different compounds over time in the continuous treatment of AuNPs (10 μg / mL) in MDRK.p of the present invention; in the graph, A is adenine, B is serine, C is isoleucine, D is homoserine, E is arginine, F is γ-aminobutyric acid, G is methionine, H is putrescine, I is spermidine, and J is spermine. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0040] Example 1

[0041] (1) In a round-bottom flask, 0.05 mmol of p-aminophenylboronic acid ABA (molecular weight 136.94, Sigma), 0.05 mmol of chloroauric acid trihydrate (molecular weight 393.83, Sinopharm Chemical Reagent Co., Ltd.), 50 μL of triethylamine, and 30 mg of Tween 80 were dissolved in 10 mL of deionized water. 6 mg of sodium borohydride was dissolved in 2 mL of deionized water, and 1 mL was added dropwise to the round-bottom flask with rapid stirring (1000 rpm). The color of the solution in the flask turned brown, and the entire reaction was carried out in an ice-water bath for 2 h.

[0042] (2) The obtained p-aminophenylboronic acid-modified gold nanoparticles (A-AuNPs) were dialyzed for 24 h using a dialysis bag (14 kDa MW cut-off, Solarbio) to remove unreacted chemicals, filtered through a 0.22 μm filter (Millipore), sterilized, and stored in a refrigerator at 4°C.

[0043] (3) The antibacterial activity of A-AuNPs was determined by the minimum inhibitory concentration (MIC). Klebsiella pneumoniae (Kp), drug-resistant Klebsiella pneumoniae (MDRK.p), Staphylococcus aureus (Sa), and methicillin-resistant Staphylococcus aureus (MRSA) were inoculated into liquid LB medium, mixed, and cultured on a shaker (260 rpm, 30°C) for 4 h. 4 CFU / mL was inoculated in liquid LB medium. A-AuNPs were diluted 2, 4, 8, 16, 32, 64, and 128 times and added to the culture medium containing bacteria. After 24 hours, the turbidity of the bacterial suspension was tested at 600 nm by the optical density (OD 600nm ) Record MIC, data such as Figure 1 As shown in A. The results showed that A-AuNPs had antibacterial activity against Gram-negative bacteria, and its MIC value for Kp and MDRK.p was 6 μg / mL, while A-AuNPs had no antibacterial activity against Gram-positive bacteria, and its MIC value for Sa and MRSA exceeded 64 μg / mL.

[0044] (4) To further observe the damage of A-AuNPs to bacteria, we placed the bacteria and A-AuNPs on a shaker at 260 rpm for 4 h. Untreated bacteria served as the control group. The bacteria were fixed, dehydrated, and observed by scanning electron microscopy. The results are shown in Figure 2. Figure 1 As shown in B. The results show that A-AuNPs can destroy the structure of bacteria, and the white arrows indicate the rupture of the bacterial cell wall.

[0045] Example 2

[0046] Here are the steps:

[0047] (1) In a round-bottom flask, 0.05 mmol of chloroauric acid trihydrate (molecular weight 393.83, Sinopharm Chemical Reagent Co., Ltd.), 50 μL of triethylamine, and 30 mg of Tween 80 were dissolved in 10 mL of deionized water. 6 mg of sodium borohydride was dissolved in 2 mL of deionized water, and 1 mL was added dropwise to the round-bottom flask with rapid stirring (1000 rpm). The color of the solution in the flask immediately turned brown, and the entire reaction was carried out in an ice-water bath for 2 h.

[0048] (2) The purification of unmodified gold nanoparticles (AuNPs) was the same as in Example 1.

[0049] (3) The antibacterial properties of AuNPs were characterized in the same way as in Example 1. Figure 1 As shown in A. The results showed that AuNPs had no antibacterial activity against both Gram-negative and Gram-positive bacteria.

[0050] Example 3

[0051] Here are the steps:

[0052] (1) Experimental use Figure 2 In the ND-EESI device shown, the angle between the sample channel and the extractant channel is 60°, the distance between the two is 2 mm, the angle between the extractant channel port and the mass spectrometer port is 150°, the angle between the sample channel port and the mass spectrometer port is 150°, and the distance b between the capillary tip and the mass spectrometer port is 4 mm. The capillary voltage and lens voltage use the system default parameters, which are 35V and 110V respectively. The analyte is detected in the positive ion mode, and the range of each parameter is as follows: the ion source uses pure water (100%), methanol: water = 1: 1 (v / v), methanol: water = 9: 1 (v / v), methanol: water: acetic acid = 47.5: 47.5: 5 (v / v / v), and methanol (100%) as the extractant, respectively. The extraction solvent passes through the quartz capillary at a flow rate of 2-14 μL / min, the capillary temperature is 150-220°C, N2 is used as the spray gas flow, and the spray gas pressure is 0.1-0.7MPa. A positive high voltage of 2-5 kV is applied to the steel needle of the microinjection needle. Under the action of the electric field, a large number of tiny charged droplets carrying the analyte are generated at the front end of the sample. These tiny charged droplets are desolvated to obtain gaseous analyte ions, which are introduced into the mass spectrometer for detection. The scanning range of the mass spectrometer is m / z 50-1000. The optimization results are as follows: Figure 3-4 The results show that when the capillary temperature is 200℃, the voltage is 4kV, the nitrogen pressure is 0.5MPa, the extractant is methanol, and the flow rate of the extractant is 10μL / min, the mass spectrum peak intensity is the highest and the collection signal is optimal.

[0053] (2) The Kp cultured to the logarithmic phase was centrifuged at 8000 rpm for 3 min and then diluted to 10 with LB medium. 5 CFU / mL. 10 μg / mL of antibacterial gold nanoparticles (A-AuNPs) were added to the bacterial solution, and the control group was added with an equal amount of LB medium. After 3.5 h of culture, the samples were centrifuged at 8000 rpm and 4 °C, and the supernatant was collected for mass spectrometry data collection.

[0054] (3) The mass spectrometry data were processed using the Xcalibur data processing system developed by Thermo Scientific. After background removal, the m / z values ​​were imported into Excel. The independent variable was the m / z value, and the dependent variable was the relative intensity of the mass spectrum. Matlab R2016a (version 9.0) released by Mathworks was used to normalize the data and perform principal component analysis (PCA). The results are shown in Figure 2. Figure 5A. The PCA results showed that there were significant differences in composition between the control group (Kp) and the experimental group (AK.p), proving that the two groups of data were significantly different. Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed using Simca software (version 14.0) from Umetrics, Sweden, to establish a model between metabolite expression and sample category for predicting sample category. The results are shown in Figure 5 B. The results show that in the supervised mode, the Kp and AK.p groups of samples show obvious differences in characteristic substances. By calculating the projection variable importance (VIP) value, the contribution of each compound to the classification is evaluated, and VIP greater than 1 is used as the screening standard. The results are shown in Figure 5 C. In the VIP diagram, the VIP values ​​of 10 differential compounds (adenine, serine, homoserine, isoleucine, putrescine, γ-aminobutyric acid, arginine, spermidine, methionine, and spermine) were greater than 1, proving that the 10 screened compounds had an important influence in explaining the variability in the model and had the potential to become key biomarkers.

[0055] Example 4

[0056] Here are the steps:

[0057] (1) The mass spectrometer apparatus and parameter optimization are the same as those in Example 3.

[0058] (2) The MDRK.p cultured to the logarithmic phase was centrifuged at 8000 rpm for 3 min and then diluted to 10 with LB medium. 5 CFU / mL. 10 μg / mL of antibacterial gold nanoparticles (A-AuNPs) were added to the bacterial solution, and the control group was added with an equal amount of LB medium. After 3.5 h of culture, the samples were centrifuged at 8000 rpm and 4 °C, and the supernatant was collected for mass spectrometry data collection.

[0059] (3) Mass spectrometry data analysis was the same as in Example 3. The results are as follows: Figure 6 The PCA results showed that there were significant differences in composition between the control group (MDRK.p) and the experimental group (A-MDRK.p), proving that the two groups of data were significantly different ( Figure 6 A). In the OPLS-DA analysis, under the supervised mode, the two groups of samples, MDRK.p and A-MDRK.p, showed obvious differences in characteristic substances ( Figure 6 B). In the VIP diagram, the VIP values ​​of the 10 differential compounds are greater than 1, proving that the 10 screened compounds have an important influence in explaining the variability in the model and have the potential to become key biomarkers ( Figure 6 C).

[0060] Example 5

[0061] Here are the steps:

[0062] (1) Combined with the mass spectral fingerprint database information of primary and secondary collision-induced dissociation (CID), the corresponding 10 compounds were predicted. In the collision-induced dissociation (CID) experiment, the parent ion isolation width was 1.6-2.0 Da, the collision energy was 20%-40%, and other conditions were automatically optimized by the system.

[0063] (2) The 10 predicted standards purchased were prepared into 10 μg / mL standard solutions. Secondary collision-induced dissociation experiments were performed under the same mass spectrometry conditions. The compounds were confirmed by comparing the mass spectrometry fingerprints of the standards and bacterial samples. The results are as follows: Figure 7 The results showed that the CID mass spectrometry fingerprints of the bacterial sample and the standard were consistent, proving the compounds corresponding to the 10 differential ions.

[0064] (3) By preparing standard solutions of different concentrations, the concentration range is 0.1-10 μg / mL, and a standard curve between the standard concentration and the relative intensity of the mass spectrometry peak is established. Using the same test conditions, the mass spectrometry peak of the bacteria at different concentrations is tested, and the bacterial concentration range is 10-10 6 CFU / mL, draw a standard curve between the relative intensity of the mass spectrometer peak and the bacterial concentration. The linear relationship between the bacterial concentration and the standard concentration is analyzed by measuring the relative intensity of the peak. The results are as follows Figure 8 The results show that there is a clear linear relationship between bacterial concentration and differential compound concentration. Specifically, when the bacterial concentration is between 10-10 6 When the CFU / mL range changes, the concentration of the corresponding compound changes linearly in the range of 0.1-200 μg / mL.

[0065] Example 6

[0066] Here are the steps:

[0067] (1) The Kp cultured to the logarithmic phase was centrifuged at 8000 rpm for 3 min and then diluted to 10 with LB medium. 5 CFU / mL.

[0068] (2) The prepared bacterial solution was added to the sample pool. In the positive ion mode, the changes of the 10 compounds were observed in real time for 210 min. Antibacterial A-AuNPs (10 μg / mL) or non-antibacterial AuNPs (10 μg / mL) were added to the sample pool at 210 min. The changes of the 10 compounds were continued to be observed. The effects of the antibacterial properties on the changes in the contents of the 10 compounds were compared. The results are shown in Figure 2. Fig. 9 and Fig.10The results show that in the first 210 minutes, the concentrations of the 10 compounds increased with time, proving the stable growth of bacteria. At 210 minutes, antibacterial A-AuNPs (10 μg / mL) were added, and the concentrations of the compounds decreased rapidly with time ( Fig. 9 ), while the addition of non-antibacterial AuNPs (10 μg / mL) did not significantly change the compound concentration, proving that A-AuNPs have a significant effect on bacterial proliferation ( Fig.10 ).

[0069] (3) The prepared bacterial solution was treated with A-AuNPs (10 μg / mL) for 210 min to obtain damaged bacteria. The above 10 substances can be divided into 3 categories. One representative substance is selected from each category. The 3 selected substances or a mixture of the 3 substances are added to the damaged bacteria respectively. The OD value is calculated. 600nm The growth of bacteria at different times was tested, and normal bacteria without A-ANPs treatment were used as controls. The results are shown in Fig.11 The results show that the growth of bacteria was effectively restored by adding the above substances to the damaged bacteria, and the overall bacterial concentration showed an increase, proving that the single component or mixed component of the above substances can repair the bacteria damaged by gold nanoparticles (A-AuNPs), indicating that A-AuNPs achieves its antibacterial properties by interfering with the above substances.

[0070] Example 7

[0071] Here are the steps:

[0072] (1) MDRK.p cultured to the logarithmic phase was centrifuged at 8000 rpm for 3 min and then diluted to 10 with LB medium. 5 CFU / mL.

[0073] (2) Mass spectrometry and data analysis were performed in the same manner as in Example 6. Fig.12 and Fig.13 The results show that in the first 210 minutes, the concentrations of the 10 compounds increased with time, proving the stable growth of bacteria. At 210 minutes, antibacterial A-AuNPs (10 μg / mL) were added, and the concentrations of the compounds decreased rapidly with time ( Fig.12 ), while the addition of non-antibacterial AuNPs (10 μg / mL) did not significantly change the compound concentration, proving that A-AuNPs have a significant effect on bacterial proliferation ( Fig.13 ).

[0074] (3) The exogenous substance supplementation experiment was the same as in Example 6. The results are as follows Fig.11As shown, the results indicate that by adding representative substances to the damaged MDRK.p, the growth of MDRK.p was effectively restored, and the overall bacterial concentration showed an increase, proving that the supplementation of the above substances, either single or mixed, had a repairing effect on bacteria damaged by gold nanoparticles (A-AuNPs), indicating that A-AuNPs achieve antibacterial properties by interfering with the above substances.

[0075] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0076] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS, characterized in that: The following steps are involved: S1, preparing antibacterial gold nanoparticles, and determining the minimum inhibitory concentration of the antibacterial gold nanoparticles to the bacteria to be tested; S2, preparing the bacterial solution to be tested; S3, adding the antibacterial gold nanoparticles to the bacterial solution to be tested, culturing for 3-4 hours, centrifuging and collecting the supernatant to obtain a test sample, wherein the concentration of the antibacterial gold nanoparticles in the bacterial solution is ≥ the minimum inhibitory concentration; centrifuging the bacterial solution to be tested that has not been treated with antibacterial gold nanoparticles, collecting the supernatant, and obtaining a control sample; performing neutral desorption electrospray extraction ionization mass spectrometry detection on the test sample and the control sample; S4, Neutral desorption electrospray extraction ionization mass spectrometry detection: The ion source of the neutral desorption electrospray extraction ionization mass spectrometer was set to positive ion mode, and the parameters were as follows: methanol was used as the extractant in the ion source, and the extractant passed through a quartz capillary, and N2 was used as the spray gas flow; a positive high voltage of 2-5 kV was applied to the steel needle of the injection needle, and under the action of the electric field, charged droplets carrying the analyte were generated at the front end of the sample, and the charged droplets were desolvated to obtain gaseous analyte ions, which were introduced into the mass spectrometer for detection; in the collision-induced dissociation experiment, the parent ion isolation width was 1.6-2.0 Da, the collision energy was 20%-40%, and other conditions were automatically optimized by the system; S5, quantitatively analyzing the differential metabolites in the test sample relative to the control sample, and verifying whether the differential metabolites are the key antibacterial targets of the antibacterial gold nanoparticles by adding the differential metabolites to the test bacterial solution containing the antibacterial gold nanoparticles; The bacteria to be tested include Klebsiella pneumoniae; The differential metabolites include one or more of adenine, serine, homoserine, isoleucine, putrescine, γ-aminobutyric acid, arginine, spermidine, methionine and spermine.

2. The method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS according to claim 1, characterized in that: The antibacterial gold nanoparticles are gold nanoparticles modified with p-aminophenylboronic acid, and the preparation method thereof is as follows: Dissolve p-aminophenylboronic acid, chloroauric acid trihydrate, triethylamine and Tween 80 in deionized water and mix well to obtain a mixed solution; Then, sodium borohydride is dissolved in deionized water to obtain a sodium borohydride solution; the sodium borohydride solution is then added dropwise to the mixed solution, stirred, and when the color of the solution in the bottle turns brown, reacted in an ice water bath for 2-2.5 hours to obtain the antibacterial gold nanoparticles.

3. The method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS according to claim 2, characterized in that: The mass volume ratio of the deionized water, p-aminophenylboronic acid, chloroauric acid trihydrate, triethylamine and Tween 80 is 8-12 mL: 6-8 mg: 18-20 mg: 45-55 μL: 26-32 mg.

4. The method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS according to claim 3, characterized in that: The mass volume ratio of the deionized water and sodium borohydride is 1-3 mL: 5-9 mg.

5. The method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS according to claim 1, characterized in that: The flow rate of the extractant was 9-12 mL / min.

6. The method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS according to claim 1, characterized in that: The spray pressure is 0.5-0.6 MPa.

7. The method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS according to claim 1, characterized in that: The temperature of the quartz capillary is 190-210 °C.

8. The method for real-time detection of the interaction between antibacterial gold nanoparticles and bacteria by ND-EESI-MS according to claim 1, characterized in that: The mass spectrometer scan range is m / z 50-1000.

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